SUMMARY/ABSTRACT Combination antiretroviral therapy (cART) can control but not cure HIV infection because of persisting viral reservoirs from which infection quickly rebounds if treatment is interrupted (TI). These reservoirs therefore must be eliminated to eradicate infection or permanently controlled to achieve a functional cure (long-term drug-free remission). Most current cure strategies are focused on the latently infected CD4 T cell reservoir, but there are other potentially important sources of virus to fuel recrudescent infection: 1) low level replication in lymphoid tissues (LT) where levels of cART drug are suboptimal for full suppression of virus replication in some treated patients; and 2) follicular CD4 T helper cells (Tfh) that persistently produce viruses in B cell follicles that provide an immunologically privileged site to escape CD8 CTL elimination. These other sources of rebound have not been sufficiently studied to date to evaluate their potential contribution to rebound or response to interventions aimed at cure. It is important to do so, since cure strategies will be quite different depending on the sources of rebound. If reactivating latently infected cells represents the likely major source of rebound, then the current focus on shock and kill strategies is appropriate; but, if cells in LT that continue to produce virus under suboptimal drug levels are the major immediate source of virus to reignite infection, then the appropriate focus to delay rebound would be better drugs and treatments for full suppression of replication in LT. This proposal describes single-cell technologies [in situ hybridization/tyramide signal amplification/immunohistochemical staining (ISH/TSA/IHC)] that enable identification, enumeration and categorization of virus-producing cells in LT during cART as reactivating latently infected cells; infected resting CD4 T cells that continue to produce virus under suboptimal levels of cART in LT; or Tfh. Measuring virus- producing cellular reservoirs in LT during cART will identify the sources of rebound following TI, which will be directly tested in one patient cohort where the size of virus producing cell reservoirs can be correlated with the time to rebound for each patient. Identifying virus-producing cellular reservoirs during cART can also provide unequivocal evidence of ongoing virus production due to suboptimal drug levels in a re-examination of a patient cohort in which correlations between virus-producing cells and drug levels can be tested in individual patients. The Specific Aims of the proposal are to 1: Determine by ISH/TSA/IHC phenotyping the number and types of HIV virus-producing cell populations in LT samples from patients with undetectable plasma viral loads on cART; and 2: Determine by ISH/TSA/IHC phenotyping the number and types of HIV virus-producing cell populations in LT samples prior to TI and correlations with time to rebound. By directly and unequivocally identifying and enumerating the types of virus-producing cells that are immediate sources for recrudescent infection in LT, the proposed work will provide a rational basis to devise and gauge the success of cure strategies.